Substantial work has been carried out to create satisfactory immunological assays or probes for measurement or detection of cholesterol. The most common analytical tool for such measurement is the chemical measurement of cholesterol or the use of a cholesterol-binding substance such as filipin or other polyene antibiotics. These latter substances are usually heterogeneous and difficult and expensive to produce in quantity and do not cause activation of complement as an amplifying mechanism. Accordingly, there remains a substantial need in the art for measurement of cholesterol level in biological specimens and particularly in humans.
The determination and presence of concentration of antigenic substances in biological specimens including those associated with various disorders, increasingly rely on immunoassay techniques. Commonly in the prior art such techniques are based upon formation of a complex between the antigenic substance being assayed and an antibody or antibodies in which one or the other member of the complex may be labeled as by a radioactive element which permits this detection and/or quantitative analyses after separation of the complexed labeled antigen or antibody from uncomplexed labeled antigen or antibody. In the case of a competition immunoassay technique, the antigenic substance in a sample of fluid being tested for its presence competes with a known quantity of labeled antigen for a limited quantity of antibody binding sites. Thus, the amount of labeled antigen bound to the antibody is inversely proportional to the amount of antigen in the sample. By contrast, immunometric assays employ a labeled antibody wherein the amount of labeled antibody associated with the complex is proportional to the amount of antigenic substance in the fluid sample. Such immunometric assays are found to be well suited for detection of polyvalent antigens, that is, antigenic substances which complex with two or more antibodies at the same time.
Despite the wide use of such prior art immunometric assays, problems are encountered in their use in that the antibodies are usually relatively impure and produce false positive reactions. Also, a single antibody may be used for binding the antigen for the detection or determination, which allows for error because of the problems with impure or non-specific antibodies.
In 1975, cell fusion techniques were developed to overcome the problem of lymphocyte non-viability in cell cultures. Techniques were devised for the construction of hybridoma cell lines wherein spleen lymphocytes from an animal immunized with an antigen are fused with mouse myeloma cells. The myeloma cells are immortal cells of the immune system, the term "immortal" meaning that they will continue to grow in the culture so long as they are continuously fed. The fusion of lymphocytes with myeloma cells leads to hybrid cells which have two important properties in that they have assumed the immortal life style of the myeloma cells and produce the antibody characteristic of the lymphocyte. Once the hybridoma cells are constructed, they are cloned and grown up so that groups of cells are obtained, each of which is producing only a single monoclonal antibody.
Monoclonal antibody techniques have been applied in a wide variety of areas, including immunometric assays. U.S. Pat. No. 4,486,530 to David et al, for example, discloses and claims sandwich immunometric assay techniques for determination of the presence or concentration of antigenic substances in fluids using monoclonal antibodies.
In U.S. Pat. No. 4,471,058 to Smith et al, monoclonal antibody techniques are described for the detection and/or determination of a polyvalent antigen using at least two different monoclonal antibodies. Substantial work has been done in detection and measurement of various types of antigens.
To Applicants' knowledge, however, none of this work has extended to cholesterol technology. Insofar as Applicants are aware, the only work involving the use of such technology with respect to cholesterol is the work of Banerji et al, Biochim. Biophys. Acta, 689, 319-326, (1982.) Banerji et al was concerned with membrane lipid compositions which modulate the binding specificity of a monoclonal antibody against liposomes. This work, however, was concerned with monoclonal antibodies which react with liposomes which contain low cholesterol amounts, and none of the work suggests the use of monoclonal antibodies which will react with high cholesterol liposomes. The present invention provides monoclonal antibody compositions and methods for detection and measurement of high cholesterol levels.